2.1. IN VITRO REGENERATION OF CHRYSANTHEMUM
The chrysanthemum, Chrysanthemum x hortorum (Bailey) L. H., Compositae, alternatively known as Chrysanthemum moripholium (Ramat) or Dendranthema grandiflora (Tzvelev is one of the most important cut flower crops (Kaul et al., 1990, Plant Cell, Tissue and Organ Culture 21:21-30). New cultivars of chrysanthemum have primarily been generated by crossbreeding (see for example VandenBerg, U.S. Pat. No. 4,827,061, issued May 2, 1989) or by mutation breeding (see for example Sparkes, U.S. Pat. No. 4,616,099, issued Oct. 7, 1986 and Broertjes et al., 1976, Euphytica 25:11-19). However, such procedures are tedious and may be unpredictable. Additionally, it may be difficult to determine the genotype of a cultivar generated by these methods.
It would be desirable to use recombinant DNA technology to confer characteristics such as new color, increased flower life, and disease and insect resistance to plants reproducibly and efficiently. However, in order to use such recombinant DNA technology, it is necessary that there be methods for transferring exogenous DNA fragments into a chrysanthemum plant. As will be described in further detail in Section 2.2. infra, attempts to transform chrysanthemum plants with exogenous DNA fragments using Agrobacterium as a vector have had only limited success. There must also be methods for efficiently culturing chrysanthemum plants in vitro. As will be discussed below, in vitro shoot multiplication, adventitious regeneration in chrysanthemum, and regeneration from cultured protoplasts have been reported by several groups.
Chrysanthemum regeneration was initially achieved using a callus intermediary (Hill, 1968, Physiol. Plant. 21:386-389). Explants of a chrysanthemum stem and receptacle were cultured on medium containing 2 mg/l naphthalene acetic acid (NAA) and 0.8 g/l kinetin and then subcultured on medium containing 1 mg/l kinetin, resulting in the production of callus and the ultimate production of shoot formation. Earle and Langhans (1974, J. Amer. Soc. Hort. Sci. 99:128-132) obtained single or multiple shoots and leafy basal callus in shoot tip culture using medium containing 2 mg/l kinetin and 0.02 mg/l NAA. Bush et al. (1976, Amer. J. Bot. 63:729-737) obtained plant regeneration from petal derived callus using medium containing 1 mg/l 6-benzyladenine (6-BA) and 1 mg/l indole acetic acid (IAA). Sangwan and Harada (1977, Acta Hortic. 78:237-242) placed various explants on media containing 2,4-dichlorophenoxy acetic acid (2,4-D) . The calli produced were used to initiate suspension cultures which they then regenerated.
Culturing of a chrysanthemum plant using a callus intermediary, however, may result in unexpected variations. For example, regeneration through a callus phase has resulted in changes in flower color and/or shape in some chrysanthemum cultivars (Khalid et al., 1989, Scientia Horticulturae 38:287-294 and Bush et al., 1976, Amer. J. Bot. 63:729-737). Abnormalities such as aberrant forms, proliferation of apical buds, variable leaf shapes and stunted growth have also been observed in plants regenerated from long term callus cultures (Sutter and Langhans, 1981, Ann. Bot. 48:559-568).
Methods have also been developed for directly producing shoots from explant tissue. Such procedures may help retain clonal fidelity. Roest and Bokelmann (1975, Scientia Horticulturae 3:317-330) reported the development of shoots using pedicels from chrysanthemum cultivars (Super Yellow, Bravo). The Super Yellow was cultivated in medium containing 1 mg/l 6-BA and 0.01 mg/l IAA. The Bravo cultivar was incubated in medium containing 1 mg/l 6-BA and 1 mg/l IAA. Recently, shoot regeneration from stem and leaf explants have been observed (Kaul et al., 1990, Plant Cell, Tissue and Organ Culture 21:21-30 and Lu et al., 1990, Plant Cell Reports 8:733-736). Both sets of investigators found that optimum results were achieved using 6-BA and NAA.
A third approach involves the use of chrysanthemum mesophyll protoplasts (Sauvadet et al., 1990, Plant Cell Reports 8:692-695). The best results were observed using medium that did not contain ammonium sulfate. Twenty-nine clones were studied. Calli were observed in five of the clones. Complete regeneration was observed in one of the clones.